Fatty Acid Incorporation in the Muscle, Oxidative Markers, Lipid Peroxidation and PPAR-α and SREBP-2 Expression of Zebrafish Fed Linseed Oil and Clove Leaf Essential Oil.

The objective of this study is to assess, in zebrafish, the effects of combining linseed oil (LO) and clove leaf essential oil (CLEO) on the incorporation of fatty acids in the muscle, oxidative markers, lipid peroxidation and expression of the PPAR-α (Peroxisome Proliferator-Activated Receptor-α) and the SREBP-2 (Sterol Regulatory Element Binding Protein-2) genes. Six diets were prepared, containing combinations of LO (3, 6 and 9%) and CLEO (0.5 and 1%): 3% LO + 0.5% CLEO; 3% LO + 1% CLEO; 6% LO + 0.5% CLEO; 6% LO + 1% CLEO; 9% LO + 0.5% CLEO; 9% LO + 1% CLEO. Results showed increase in the incorporation of n-3 fatty acids in the muscle concomitantly with the addition of LO and CLEO. The activities of superoxide dismutase and catalase were reduced and the glutathione content had increased. Lipid peroxidation was lower in the treatment with 1% CLEO, regardless of LO content. The expression of the PPAR-α and the SREBP-2 genes was higher in animals fed 9% LO + 0.5% CLEO. Therefore, for a greater incorporation and protection against the oxidative damages of n-3 fatty acids, a combined use of 9% LO with 0.5% CLEO is recommended for zebrafish.

Enhanced cytotoxicity of imidacloprid by biotransformation in isolated hepatocytes and perfused rat liver.

Imidacloprid (IMD) is a neonicotinoid insecticide widely used in crops, pets, and on farm animals for pest control, which can cause hepatotoxicity in animals and humans. In a previous study using isolated rat liver mitochondria, we observed that IMD inhibited the activity of FoF1-ATP synthase. The aim of this study was to evaluate the effects of IMD on rat isolated hepatocytes and perfused rat liver, besides the influence of its biotransformation on the toxicological potential. For the latter goal, rats were pretreated with dexamethasone or phenobarbital, two classical cytochrome P-450 stimulators, before hepatocytes isolation or liver perfusion. IMD (150 and 200 µM) reduced state 3 mitochondrial respiration in digitonin-permeabilized cells that were energized with glutamate plus malate but did not dissipate the mitochondrial membrane potential. In intact (non-permeabilized) hepatocytes, the intracellular ATP concentration and cell viability were reduced when high IMD concentrations were used (1.5-3.0 mM), and only in cells isolated from dexamethasone-pretreated rats, revealing that IMD biotransformation increases its toxicity and that IMD itself affects isolated mitochondria or mitochondria in permeabilized hepatocytes in concentrations that do not affect mitochondrial function in intact hepatocytes. Coherently, in the prefused liver, IMD (150 and 250 µM) inhibited gluconeogenesis from alanine, but without affecting oxygen consumption and urea production, indicating that such effect was not of mitochondrial origin. The gluconeogenesis inhibition was incomplete and occurred only when the rats were pretreated with phenobarbital, signs that IMD biotransformation was involved in the observed effect. Our findings reveal that changes in hepatic energy metabolism may be acutely implicated in the hepatotoxicity of IMD only when animals and humans are exposed to high levels of this compound, and that IMD metabolites seem to be the main cause for its toxicity.

ATP11C targets basolateral bile salt transporter proteins in mouse central hepatocytes.

UNLABELLED: ATP11C is a homolog of ATP8B1, both of which catalyze the transport of phospholipids in biological membranes. Mutations in ATP8B1 cause progressive familial intrahepatic cholestasis type1 in humans, which is characterized by a canalicular cholestasis. Mice deficient in ATP11C are characterized by a conjugated hyperbilirubinemia and an unconjugated hypercholanemia. Here, we have studied the hypothesis that ATP11C deficiency interferes with basolateral uptake of unconjugated bile salts, a process mediated by organic anion-transporting polypeptide (OATP) 1B2. ATP11C localized to the basolateral membrane of central hepatocytes in the liver lobule of control mice. In ATP11C-deficient mice, plasma total bilirubin levels were 6-fold increased, compared to control, of which ∼65% was conjugated and ∼35% unconjugated. Plasma total bile salts were 10-fold increased and were mostly present as unconjugated species. Functional studies in ATP11C-deficient mice indicated that hepatic uptake of unconjugated bile salts was strongly impaired whereas uptake of conjugated bile salts was unaffected. Western blotting and immunofluorescence analysis demonstrated near absence of basolateral bile salt uptake transporters OATP1B2, OATP1A1, OATP1A4, and Na(+) -taurocholate-cotransporting polypeptide only in central hepatocytes of ATP11C-deficient liver. In vivo application of the proteasome inhibitor, bortezomib, partially restored expression of these proteins, but not their localization. Furthermore, we observed post-translational down-regulation of ATP11C protein in livers from cholestatic mice, which coincided with reduced OATP1B2 levels. CONCLUSIONS: ATP11C is essential for basolateral membrane localization of multiple bile salt transport proteins in central hepatocytes and may act as a gatekeeper to prevent hepatic bile salt overload. Conjugated hyperbilirubinemia and unconjugated hypercholanemia and loss of OATP expression in ATP11C-deficient liver strongly resemble the characteristics of Rotor syndrome, suggesting that mutations in ATP11C can predispose to Rotor syndrome. (Hepatology 2016;64:161-174).